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Abstract

Co-immunoprecipitation assay of TLR3-Flag or Myc-MSR1 with HCV RNA is used to identify direct interaction of viral RNA with host proteins that recognize viral RNA to initiate interferon (IFN) signaling, a crucial antiviral response of the host cells. Both Toll-like receptor 3 (TLR3) and class-A scavenger receptor type 1 (MSR1) proteins recognize viral double-stranded RNA (dsRNA) which may be released into the extracellular milieu or spread from HCV-infected cells to uninfected neighbor cells via cell-to-cell contact, resulting in IFN-β activation that restricts viral propagation. We have found that MSR1 binds extracellular dsRNA, mediating its endocytosis and transport toward the endosome where it is engaged by TLR3, thereby triggering IFN responses in both infected and uninfected cells. We used this assay to demonstrate the pivotal role of MSR1 in mediating TLR3-recognition of the HCV RNA. The assay described in this protocol is based on the conventional protein immunoprecipitation protocol with conditioned buffers that prevent nonspecific RNA degradation by RNase present in the lysate. RNA molecules associated with the Flag-tagged protein were trapped by a specific antibody followed by Protein G capture, extracted and detected quantitatively by RT-PCR assay, followed by agarose-gel electrophoresis for visualization. This method can also be applied to detection of other protein-RNA interactions.

Suspend the sepharose in 100 μl DPBS and use half for RNA extraction and the remainder for Western blotting to detect the immunoprecipitated protein.

RNA extraction and RT-PCR

Extract RNA from the sepharose beads by vortexing 15 sec in TRIzol reagent, followed by the standard protocol as indicated in the manufacturer’s instruction, and suspend the RNA pellet in 50 μl of nuclease-free water (optional: Add 1 μl of Glycogen before the precipitation of RNA with Isopropanol).

This work was supported in part by grants from the National Institutes of Health (RO1-AI095690) and the University Cancer Research Fund. This protocol is adapted from previous work by Dansako et al. (2013).

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